Whole-body optical imaging of gene expression and uses thereof

ABSTRACT

The invention relates to the whole-body external optical imaging of gene expression. Specifically, methods for whole-body external optical imaging of gene expression and methods for evaluating a candidate protocol or drug for treating diseases or disorders using a fluorophore operatively linked to the promoter of a gene and external optical imaging are provided herein. Methods to screen for substances or genes that regulate target promoters are also provided.

[0001] This application claims priority under 35 U.S.C. 119 fromprovisional application U.S. Serial No. 60/190,196 filed 17 Mar. 2000,the contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The invention relates to the whole-body external optical imagingof gene expression. Specifically, methods for whole-body externaloptical imaging of gene expression and methods for evaluating acandidate protocol or drug for treating diseases or disorders using afluorophore operatively linked to the promoter of a gene and externaloptical imaging are provided herein. Methods to screen for substances orgenes that regulate target promoters are also provided.

BACKGROUND ART

[0003] Whole-body imaging technology has been used to monitor “tracermolecules” in the intact body. For example, Brenner et al. studied thediagnostic value ofiodine-123-2-hydroxy-3-iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide (IBZM) whole-body imaging in comparison to thallium-201scintigraphy in patients with metastatic malignant melanoma (Brenner etal., Eur. J. Nucl. Med., 26(12):1567-71 (1999)). Benard et al. conductedclinical evaluation of processing techniques for attenuation correctionwith ¹³⁷Cs in whole-body PET imaging (Benard et al., J. Nucl. Med.,40(8):1257-63 (1999)). Jerusalem et al. showed that whole-body positronemission tomography using ¹⁸F-fluorodeoxyglucose for posttreatmentevaluation in Hodgkin's disease and non-Hodgkin's lymphoma has higherdiagnostic and prognostic value than classical computed tomography scanimaging (Jerusalem et al., Blood, 94(2):429-33 (1999)). Eustace et al.discussed practical issues, clinical applications, and future directionsof whole-body MR imaging (Eustace et al., Magn. Reson. Imaging Clin. (N.Am), 7(2):209-36 (1999)). Engelson et al. studied fat distribution inHIV-infected patients reporting truncal enlargement quantified bywhole-body magnetic resonance imaging (Engelson et al., Am. J. Clin.Nutr., 69(6):1162-9 (1999)). Valk et al. used whole-body positronemission tomography (PET) imaging with [¹⁸F]fluorodeoxyglucose inmanagement of recurrent colorectal cancer (Valk et al., Arch. Surg.,134(5):503-11 (1999)). Saunders et al. evaluatedfluorine-18-fluorodeoxyglucose whole body positron emission tomographyimaging in the staging of lung cancer (Saunders et al., Ann. Thorac.Surg., 67(3):790-7 (1999)).

[0004] U.S. Pat. No. 5,650,135 discloses a noninvasive method fordetecting the localization of an entity under study from within amammalian subject, which method comprises: (a) administering to thesubject a conjugate of the entity and a light-generating moiety or atransformed cell expressing the light-generating moiety; (b) after aperiod of time in which the conjugate or transformed cell can achievelocalization in the subject, immobilizing the subject within thedetection field of a photodetector device; (c) maintaining the subjectin an immobilized condition, (d) during said maintaining, measuringphoton emission from the light-generating moiety, localized in thesubject, with the photodetector device until an image of photon emissioncan be constructed; and (e) detecting said image through an opaquetissue of said mammal. U.S. Pat. No. 5,650,135 also discloses anoninvasive method for detecting the level of an entity under study in amammalian subject over time, which method comprises: (a) administeringto the subject a conjugate of the entity and a light-generating moietyor a transformed cell expressing the light-generating moiety; (b)placing the subject within the detection field of a photodetectordevice; (c) maintaining the subject in the detection field of thedevice; (d) during said maintaining, measuring photon emission from thelight-generating moiety, in the subject, with the photodetector device;and (e) repeating steps (b) through (d) at selected intervals, whereinsaid repeating is effective to detect changes in the level of the entityin the subject over time.

[0005] Recently, Yang et al. conducted whole-body optical imaging ofgreen fluorescent protein-expressing tumors and metastases (Yang et al.,Proc. Natl. Acad. Sci. (USA), 97(3):1206-11 (2000)). Yang et al. haveimaged, in real time, fluorescent tumors growing and metastasizing inlive mice. The whole-body optical imaging system is external andnoninvasive. It affords unprecedented continuous visual monitoring ofmalignant growth and spread within intact animals. Yang et al. haveestablished new human and rodent tumors that stably express very highlevels of the Aequorea victoria green fluorescent protein (GFP) andtransplanted these to appropriate animals. B16F0-GFP mouse melanomacells were injected into the tail vein or portal vein of 6-week-oldC57BL/6 and nude mice. Whole-body optical images showed metastaticlesions in the brain, liver, and bone of B16F0-GFP that were used forreal time, quantitative measurement of tumor growth in each of theseorgans. The AC3488-GFP human colon cancer was surgically implantedorthotopically into nude mice. Whole-body optical images showed, in realtime, growth of the primary colon tumor and its metastatic lesions inthe liver and skeleton. Imaging was with either a trans-illuminatedepifluorescence microscope or a fluorescence light box andthermoelectrically cooled color charge-coupled device camera. The depthto which metastasis and micrometastasis could be imaged depended ontheir size. A 60-micrometer diameter tumor was detectable at a depth of0.5 mm whereas a 1, 800-micrometer tumor could be visualized at 2.2-mmdepth. The simple, noninvasive, and highly selective imaging of growingtumors, made possible by strong GFP fluorescence, enables the detailedimaging of tumor growth and metastasis formation. This should facilitatestudies of modulators of cancer growth including inhibition by potentialchemotherapeutic agents.

[0006] Methods for monitoring gene expression are known in the art (seegenerally, Ausubel et al. (Ed.), Current Protocols in Molecular Biology,John Wiley & Sons, Inc.). However, whole-body external optical imagingof gene expression, which offers simple, noninvasive, highly selective,and real-time recording and analysis of gene expression in an intactmulti-cellular organisms, e.g., animals, is not available currently. Thepresent invention addresses this and other related needs in the art.

DISCLOSURE OF THE INVENTION

[0007] The invention provides for whole-body external optical imaging ofgene expression and methods for evaluating a candidate protocol or drugfor treating diseases or disorders. The method uses a fluorophoreoperatively linked to the promoter of a gene and external opticalimaging. Methods to screen for substances or genes that regulate targetpromoters are also provided.

[0008] In a specific embodiment, a method to monitor the expression of agene is provided, which method comprises: a) delivering to amulti-cellular organism a nucleic acid encoding a fluorophoreoperatively linked to the promoter of a gene whose expression is to beanalyzed or delivering a cell containing said nucleic acid; and b)observing the presence, absence or intensity of the fluorescencegenerated by said fluorophore at various locations in said organism bywhole-body external fluorescent optical imaging, whereby the expressionof said gene is monitored.

[0009] In a preferred embodiment, a nucleic acid encoding a fluorophoreoperatively linked to the promoter of the gene is delivered directly tothe organism. Also preferably, the nucleic acid encoding a fluorophoreoperatively linked to the promoter of the gene is in a viral vector suchas a viral vector derived from adenovirus or a lentivirus.

[0010] In another preferred embodiment, a cell containing a nucleic acidencoding a fluorophore operatively linked to the promoter of the gene isdelivered to the organism. More preferably, the cell is delivered to theorganism via a surgical procedure such as direct implantation bysurgical orthotopic implantation (SOI) at a desired site.

[0011] In still another preferred embodiment, the fluorophoreoperatively linked to the promoter of a gene is a humanized fluorophore.Also preferably, the fluorophore is a green fluorescent protein (GFP), ablue fluorescent protein (BFP) or a red fluorescent protein (RFP). Morepreferably, the GFP is the humanized hGFP-S65T.

[0012] In yet another preferred embodiment, the multi-cellular organismto be analyzed is a plant or an animal, including a transgenic animal.More preferably, the animal is a mammal. A human can also be analyzed bythe present method.

[0013] In yet another preferred embodiment, the gene to be analyzed isexpressed in a tissue or organ specific manner. More preferably, thegene is expressed in connective, epithelium, muscle or nerve tissue.Also more preferably, the gene is expressed in an internal animal organsuch as brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, gland,internal blood vessels, etc. Yet more preferably, the gene to beanalyzed is a tumor or cancer associated gene such as an oncogene or atumor suppressor gene.

[0014] In yet another preferred embodiment, the expression of more thanone gene is monitored simultaneously.

[0015] In another specific embodiment, a method to evaluate a candidateprotocol or drug for treating a disease or disorder is provided, whichmethod comprises: a) administering said protocol or drug to a non-humanmammalian subject which expresses a fluorophore under the direction of apromoter of a gene associated with a disease or disorder, anddetermining the expression of said promoter via observing the presence,absence or intensity of the fluorescence generated by said fluorophoreat various locations in said mammalian subject by whole-body externalfluorescent optical imaging; b) determining the expression of saidpromoter, via observing the presence, absence or intensity of thefluorescence generated by said fluorophore at various locations in saidmammalian subject by whole-body external fluorescent optical imaging, ina control non-human mammalian subject which expresses said fluorophoreunder the direction of said promoter of said gene; and c) comparing theexpression of said promoter determined in steps a) and b), wherein theexpression determined in step a) is different from that in step b)identifies said protocol or drug as effective in treating the disease ordisorder.

[0016] If overexpression of the gene is associated with the disease ordisorder, the expression determined in step a) is lower than that instep b) when said protocol or drug is effective in treating the diseaseor disorder.

[0017] If underexpression of the gene is associated with the disease ordisorder, the expression determined in step a) is higher than that instep b) when said protocol or drug is effective in treating theinfection.

[0018] Preferably, the disease or disorder is a cancer, an immune systemdisease or disorder, a metabolism disease or disorder, a muscle and bonedisease or disorder, a nervous system disease or disorder, a signaldisease or disorder, or a transporter disease or disorder.

[0019] Preferably, the non-human mammalian subject which expresses afluorophore under the direction of a promoter of the gene is produced bydelivering a nucleic acid encoding the fluorophore operatively linked tothe promoter of the gene, or a cell containing the nucleic acid, to thenon-human mammalian subject. Alternatively, the non-human mammaliansubject used in the screen is a transgenic animal.

[0020] The non-human mammalian subject used in the screening ispreferably a well established laboratory animal such as a mice, a rabbitor a non-human primate.

[0021] The fluorophore used in the screening is preferably a greenfluorescent protein (GFP), a blue fluorescent protein (BFP) or a redfluorescent protein (RFP).

[0022] More than one candidate protocol or candidate drug is preferablyscreened for simultaneously.

[0023] If the non-human mammalian subject expresses a fluorophore underthe direction of a promoter of an infectious organism, the expressiondetermined in step a) is lower than that in step b) when said protocolor drug is effective in treating infection caused by the infectiousorganism.

[0024] The non-human mammalian subject used in the screening ispreferably an infectious disease animal model.

[0025] The infectious organism screened against is preferably a fungussuch as a yeast, a bacterium such as an eubacteria or an archaebacteria,or a virus such as a Class I virus, a Class II virus, a Class III virus,a Class IV virus, a Class V virus or a Class VI virus.

[0026] If the infection is caused by a bacterium, the candidate drug tobe screened is preferably an antibiotic.

[0027] In still another specific embodiment, a method to screen for amodulator of the expression of a gene in a non-human multi-cellularorganism is provided, which method comprises: a) administering a testsubstance to a non-human multi-cellular organism which expresses afluorophore under the direction of a promoter of a gene, and determiningthe expression of said promoter via observing the presence, absence orintensity of the fluorescence generated by said fluorophore at variouslocations in said multi-cellular organism by whole-body externalfluorescent optical imaging; b) determining the expression of saidpromoter, via observing the presence, absence or intensity of thefluorescence generated by said fluorophore at various locations bywhole-body external fluorescent optical imaging, in a controlmulti-cellular organism which expresses said fluorophore under thedirection of said promoter of said gene; and c) comparing the expressionof said promoter determined in steps a) and b), wherein the expressiondetermined in step a) is different from that in step b) identifies saidtest substance as a modulator of said gene expression. Preferably, thepromoter is an endogenous promoter of the multi-cellular organism.

[0028] In yet another specific embodiment, a method to screen for anon-human multi-cellular organism that expresses a gene at an alteredlevel is provided, which method comprises: a) administering amutation-inducing agent or treatment to a non-human multi-cellularorganism which expresses a fluorophore under the direction of a promoterof a gene, and determining the expression of said promoter via observingthe presence, absence or intensity of the fluorescence generated by saidfluorophore at various locations in said multi-cellular organism bywhole-body external fluorescent optical imaging; b) determining theexpression of said promoter, via observing the presence, absence orintensity of the fluorescence generated by said fluorophore at variouslocations by whole-body external fluorescent optical imaging in anuntreated control multi-cellular organism which expresses saidfluorophore under the direction of said promoter of said gene; and c)comparing the expression of said promoter determined in steps a) and b),wherein the expression determined in step a) is different from that instep b) identifies a multi-cellular organism that expresses said gene atthe altered level. Preferably, the mutation-inducing agent or treatmentcauses a mutation in germ-line cells of the multi-cellular organism sothat the desired mutation is stably-transferable to offspring of themulti-cellular organism.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIGS. 1A and 1B show the time course of expression ofadenoviral-administered GFP in brain and liver respectively.Fluorescence first becomes visible in the brain within six (6) hoursafter local delivery and liver fluorescence became detectable at aboutseven (7) hours after injection into the tail vein.

[0030]FIGS. 2A and 2B art pertinent to administration of lentiviralvectors. FIG. 2A is a diagram of lentiviral vector GFP-LV. FIG. 2B is adiagram of a control observation method; whole body measurement involveduse of a light box.

MODES OF CARRYING OUT THE INVENTION

[0031] A. Definitions

[0032] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one ofordinary skill in the art to which this invention belongs. All patents,applications, published applications and other publications andsequences from GenBank and other data bases referred to herein areincorporated by reference in their entirety.

[0033] As used herein, “delivering a nucleic acid to a multi-cellularorganism” refers to a process in which the nucleic acid is eitheradministered directly into the body of the multi-cellular organism, orthe nucleic acid is administered into a cell first, and then the cellcontaining the nucleic acid is administered into the body of themulti-cellular organism. After delivery into the organism, the nucleicacid may exist independently from the genome of the host organism or maybe integrated into the genome of the host organism. If the nucleic acidis integrated into a germline cell of the host organism, such nucleicacid may be transmitted into the host organism's offspring.

[0034] As used herein, “whole-body external fluorescent optical imaging”refers to an imaging process in which the presence, absence or intensityof the fluorescence generated by the fluorophore at various locations inthe host organism is monitored, recorded and/or analyzed externallywithout any procedure, e.g., surgical procedure, to expose and/or toexcise the desired observing site from the host organism. To achieve thewhole-body external fluorescent optical imaging, it is necessary thatthe intensity of the fluorescence generated by the fluorophore issufficiently high so that, even when the fluorescence site is aninternal one within the host organism body, the fluorescence signal canbe analyzed externally without exposing or excising the site from thehost body, or while the animal is not controlled.

[0035] As no invasive procedures are required and the intensity of thesignal is sufficiently great for direct observation, the animal mayremain completely mobile and need not be restrained. The ability toprovide a completely non-invasive observation protocol is highlysignificant. If the animal is traumatized either by, e.g., incision orby physical restraint, e.g., straps or pins, the alteration inmetabolism may affect the expression of the genes in organs or tissues.

[0036] Since whole-body external fluorescent optical imaging are quickand easily amenable to automation, it can be used for monitoring largenumber of gene expression simultaneously. In addition, it can beemployed in high-throughput screening methods for identifying protocols,substances including candidate drugs, and cis-acting regulators thatregulate the expression of a target gene. Using the whole-body externalfluorescent optical imaging provided in this application, multiplecandidate protocols, substances, drugs, and cis-acting regulators can bescreened for, either against a single target gene or against multipletarget genes, in either a single animal or in multiple, animals,simultaneously.

[0037] As used herein, “fluorophore” refers to a protein that isauto-fluorescent such that no other substrates or co-factors are neededfor it to fluoresce. Non-limiting examples of such fluorophores includegreen fluorescent proteins (GFPs), blue fluorescent proteins (BFPs) andred fluorescent protein (RFPs), and functional fragments, derivativesand analogues thereof.

[0038] As used herein, “a promoter region or promoter element” refers toa segment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences that modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis actingor may be responsive to trans acting factors. Promoters, depending uponthe nature of the regulation, may be constitutive or regulated.

[0039] As used herein, “operatively linked or operationally associated”refers to the functional relationship of DNA with regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of DNA to a promoter refers tothe physical and functional relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA. In order to optimize expression and/or in vitrotranscription, it may be necessary to remove, add or alter 5′untranslated portions of the clones to eliminate extra, potentialinappropriate alternative translation initiation (i.e., start) codons orother sequences that may interfere with or reduce expression, either atthe level of transcription or translation. Alternatively, consensusribosome binding sites (see, e.g., Kozak, J. Biol. Chem.,266:19867-19870 (1991)) can be inserted immediately 5′ of the startcodon and may enhance expression. The desirability of (or need for) suchmodification may be empirically determined.

[0040] As used herein, “humanized fluorophore” refers to a fluorophorewhose codon is modified according to the codon usage pattern in humangenome to enhance its expression while substantially maintaining itsfluorescent characteristics.

[0041] As used herein, “multi-cellular organism” refers to an organismwith certain cell numbers, mass, and internal structure so that internalsites of such multi-cellular organism are not externally detectable bynon-fluorescent optical imaging without exposing the internal sites.Sufficiently high intensity of internal fluorescence is needed forexternal fluorescent optical imaging of the internal site.

[0042] As used herein, “plant” refers to any of various photosynthetic,eucaryotic multi-cellular organisms of the kingdom Plantae,characteristically producing embryos, containing chloroplasts, havingcellulose cell walls and lacking locomotion.

[0043] As used herein, “animal” refers to a multi-cellular organism ofthe kingdom of Animalia, characterized by a capacity for locomotion,nonphotosynthetic metabolism, pronounced response to stimuli, restrictedgrowth and fixed bodily structure. Non-limiting examples of animalsinclude birds such as chickens, vertebrates such fish and mammals suchas mice, rats, rabbits, cats, dogs, pigs, cows, ox, sheep, goats,horses, monkeys and other non-human primates.

[0044] As used herein, “expressed in a tissue or organ specific manner”refers to a gene expression pattern in which a gene is expressed, eithertransiently or constitutively, only in certain tissues or organs, butnot in other tissues or organs.

[0045] As used herein, “tissue” refers to a collection of similar cellsand the intracellular substances surrounding them. There are four basictissues in the body: 1) epithelium; 2) connective tissues, includingblood, bone, and cartilage; 3) muscle tissue; and 4) nerve tissue.

[0046] As used herein, “organ” refers to any part of the body exercisinga specific function, as of respiration, secretion or digestion.

[0047] As used herein, “disease or disorder” refers to a pathologicalcondition in an organism resulting from, e.g., infection or geneticdefect, and characterized by identifiable symptoms.

[0048] As used herein, neoplasm (neoplasia) refers to abnormal newgrowth, and thus means the same as tumor, which may be benign ormalignant. Unlike hyperplasia, neoplastic proliferation persists even inthe absence of the original stimulus.

[0049] As used herein, cancer refers to a general term for diseasescaused by any type of malignant tumor.

[0050] As used herein, “oncogene” refers to a mutated and/oroverexpressed version of a normal gene of animal cells (theproto-oncogene) that in a dominant fashion can release the cell fromnormal restraints on growth, and thus alone, or in concert with otherchanges, convert a cell into a tumor cell. Exemplary oncogenes include,but are not limited to, abl, erba, erbB, ets, fes (fps), fgr, fms, fos,hst, int1, int2, jun, hit, B-lym, mas, met, mil (raf), mos, myb, myc,N-myc, neu (ErbB2), ral (mil), Ha-ras, Ki-ras, N-ras, rel, ros, sis,src, ski, trk and yes.

[0051] As used herein, “tumor suppressor gene” (or anti-oncogene, cancersusceptibility gene) refers to a gene that encodes a product whichnormally negatively regulates the cell cycle, and which must be mutatedor otherwise inactivated before a cell can proceed to rapid division.Exemplary tumor suppressor genes include, but are not limited to, p16,p21, p53, RB (retinoblastoma), WT-1 (Wilm's tumor), DCC (deleted incolonic carcinoma), NF-1 (neurofibrosarcoma) and APC (adenomatouspolypospis coli).

[0052] As used herein, “an immune system disease or disorder” refers toa pathological condition caused by a defect in the immune system. Theimmune system is a complex and highly developed system, yet its missionis simple: to seek and kill invaders. If a person is born with aseverely defective immune system, death from infection by a virus,bacterium, fungus or parasite w ill occur. In severe combinedimmunodeficiency, lack of an enzyme means that toxic waste builds upinside immune system cells, killing them and thus devastating the immunesystem. A lack of immune system cells is also the basis for DiGeorgesyndrome: improper development of the thymus gland means that T cellproduction is diminished. Most other immune disorders result from eitheran excessive immune response or an ‘autoimmune attack’. For example,asthma, familial Mediterranean fever and Crohn disease (inflammatorybowel disease) all result from an over-reaction of the immune system,while autoimmune polyglandular syndrome and some facets of diabetes aredue to the immune system attacking ‘self’ cells and molecules. A keypart of the immune system's role is to differentiate between invadersand the body's own cells—when it fails to make this distinction, areaction against ‘self’ cells and molecules causes autoimmune disease.

[0053] As used herein, “a metabolism disease or disorder” refers to apathological condition caused by errors in metabolic processes.Metabolism is the means by which the body derives energy and synthesizesthe other molecules it needs from the fats, carbohydrates and proteinswe eat as food, by enzymatic reactions helped by minerals and vitamins.There is a significant level of tolerance of errors in the system:often, a mutation in one enzyme does not mean that the individual willsuffer from a disease. A number of different enzymes may compete tomodify the same molecule, and there may be more than one way to achievethe same end result for a variety of metabolic intermediates. Diseasewill only occur if a critical enzyme is disabled, or if a controlmechanism for a metabolic pathway is affected.

[0054] As used herein, “a muscle and bone disease or disorder” refers toa pathological condition caused by defects in genes important for theformation and function of muscles, and connective tissues. Connectivetissue is used herein as a broad term that includes bones, cartilage andtendons. For example, defects in fibrillin—a connective tissue proteinthat is important in making the tissue strong yet flexible—cause Marfansyndrome, while diastrophic dysplasia is caused by a defect in a sulfatetransporter found in cartilage. Two diseases that originate through adefect in the muscle cells themselves are Duchenne muscular dystrophy(DMD) and myotonic dystrophy (DM). DM is another ‘dynamic mutation’disease, similar to Huntington disease, that involves the expansion of anucleotide repeat, this time in a muscle protein kinase gene. DMDinvolves a defect in the cytoskeletal protein, dystrophin, which isimportant for maintaining cell structure.

[0055] As used herein, “a nervous system disease or disorder” refers toa pathological condition caused by defects in the nervous systemincluding the central nervous system, i.e., brain, and the peripheralnervous system. The brain and nervous system form an intricate networkof electrical signals that are responsible for coordinating muscles, thesenses, speech, memories, thought and emotion. Several diseases thatdirectly affect the nervous system have a genetic component: some aredue to a mutation in a single gene, others are proving to have a morecomplex mode of inheritance. As our understanding of the pathogenesis ofneurodegenerative disorders deepens, common themes begin to emerge:Alzheimer brain plaques and the inclusion bodies found in Parkinsondisease contain at least one common component, while Huntington disease,fragile X syndrome and spinocerebellar atrophy are all ‘dynamicmutation’ diseases in which there is an expansion of a DNA repeatsequence. Apoptosis is emerging as one of the molecular mechanismsinvoked in several neurodegenerative diseases, as are other, specific,intracellular signaling events. The biosynthesis of myelin and theregulation of cholesterol traffic also figure in Charcot-Marie-Tooth andNeimann-Pick disease, respectively.

[0056] As used herein, “a signal disease or disorder” refers to apathological condition caused by defects in the signal transductionprocess. Signal transduction within and between cells mean that they cancommunicate important information and act upon it. Hormones releasedfrom their site of synthesis carry a message to their target site, as inthe case of leptin, which is released from adipose tissue (fat cells)and transported via the blood to the brain. Here, the leptin signalsthat enough has been eaten. Leptin binds to a receptor on the surface ofhypothalamus cells, triggering subsequent intracellular signalingnetworks. Intracellular signaling defects account for several diseases,including cancers, ataxia telangiectasia and Cockayne syndrome. FaultyDNA repair mechanisms are also invoked in pathogenesis, since control ofcell division, DNA synthesis and DNA repair all are inextricably linked.The end-result of many cell signals is to alter the expression of genes(transcription) by acting on DNA-binding proteins. Some diseases are theresult of a lack of or a mutation in these proteins, which stop themfrom binding DNA in the normal way. Since signaling networks impinge onso many aspects of normal function, it is not surprising that so manydiseases have at least some basis in a signaling defect.

[0057] As used herein, “a transporter disease or disorder” refers to apathological condition caused by defects in a transporter, channel orpump. Transporters, channels or pumps that reside in cell membranes arekey to maintaining the right balance of ions in cells, and are vital fortransmitting signals from nerves to tissues. The consequences of defectsin ion channels and transporters are diverse, depending on where theyare located and what their cargo is. For example, in the heart, defectsin potassium channels do not allow proper transmission of electricalimpulses, resulting in the arrhythmia seen in long QT syndrome. In thelungs, failure of a sodium and chloride transporter found in epithelialcells leads to the congestion of cystic fibrosis, while one of the mostcommon inherited forms of deafness, Pendred syndrome, looks to beassociated with a defect in a sulphate transporter.

[0058] As used herein, “infection” refers to invasion of the body of amulti-cellular organism with organisms that have the potential to causedisease.

[0059] As used herein, “infectious organism” refers to an organism thatis capable to cause infection of a multi-cellular organism. Mostinfectious organisms are microorganisms such as viruses, bacteria andfungi.

[0060] As used herein, “bacteria” refers to small prokaryotic organisms(linear dimensions of around 1 μm) with non-compartmentalized circularDNA and ribosomes of about 70S. Bacteria protein synthesis differs fromthat of eukaryotes. Many anti-bacterial antibiotics interfere withbacteria proteins synthesis but do not affect the infected host.

[0061] As used herein, “eubacteria” refers to a major subdivision of thebacteria except the archaebacteria. Most Gram-positive bacteria,cyanobacteria, mycoplasmas, enterobacteria, pseudomonas and chloroplastsare eubacteria. The cytoplasmic membrane of eubacteria containsester-linked lipids; there is peptidoglycan in the cell wall (ifpresent); and no introns have been discovered in eubacteria.

[0062] As used herein, “archaebacteria” refers to a major subdivision ofthe bacteria except the eubacteria. There are 3 main orders ofarchaebacteria: extreme halophiles, methanogens and sulphur-dependentextreme thermophiles. Archaebacteria differs from eubacteria inribosomal structure, the possession (in some case) of introns, and otherfeatures including membrane composition.

[0063] As used herein, “virus” refers to obligate intracellularparasites of living but non-cellular nature, consisting of DNA or RNAand a protein coat. Viruses range in diameter from about 20 to about 300nm. Class I viruses (Baltimore classification) have a double-strandedDNA as their genome; Class II viruses have a single-stranded DNA astheir genome; Class III viruses have a double-stranded RNA as theirgenome; Class IV viruses have a positive single-stranded RNA as theirgenome, the genome itself acting as mRNA; Class V viruses have anegative single-stranded RNA as their genome used as a template for mRNAsynthesis; and Class VI viruses have a positive single-stranded RNAgenome but with a DNA intermediate not only in replication but also inmRNA synthesis. The majority of viruses are recognized by the diseasesthey cause in plants, animals and prokaryotes. Viruses of prokaryotesare known as bacteriophages.

[0064] As used herein, “fungi” refers to a division of eucaryoticorganisms that grow in irregular masses, without roots, stems, orleaves, and are devoid of chlorophyll or other pigments capable ofphotosynthesis. Each organism (thallus) is unicellular to filamentous,and possess branched somatic structures (hyphae) surrounded by cellwalls containing glucan or chitin or both, and containing true nuclei.

[0065] As used herein, “antibiotic” refers to a substance either derivedfrom a mold or bacterium or organically synthesized, that inhibits thegrowth of certain microorganisms without substantially harming the hostof the microorganisms to be killed or inhibited.

[0066] As used herein, “test substance” refers to a chemically definedcompound (e.g., organic molecules, inorganic molecules,organic/inorganic molecules, proteins, peptides, nucleic acids,oligonucleotides, lipids, polysaccharides, saccharides, or hybrids amongthese molecules such as glycoproteins, etc.) or mixtures of compounds(e.g., a library of test compounds, natural extracts or culturesupernatants, etc.) whose effect on the promoter to be analyzed isdetermined by the disclosed and/or claimed methods herein.

[0067] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the subsectionsthat follow.

[0068] B. Methods of Whole-Body External Optical Imaging of GeneExpression

[0069] In a specific embodiment, a method to monitor the expression of agene is provided herein, which method comprises: a) delivering to amulti-cellular organism a nucleic acid encoding a fluorophoreoperatively linked to the promoter of a gene whose expression is to beanalyzed or a cell containing said nucleic acid; and b) observing thepresence, absence or intensity of the fluorescence generated by saidfluorophore at various locations in said organism by whole-body externalfluorescent optical imaging, whereby the expression of said gene ismonitored.

[0070] The present methods can be used to monitor gene expression forany suitable purposes including prognostic, diagnostic and screeningpurposes. For example, if abnormal gene expression is associated with adisease or disorder in a multi-cellular organism such as a plant or ananimal, the present method can be used in prognosis or diagnosis bymonitoring the abnormal gene expression. The present monitoring methodsare advantageous over the currently available gene expression monitoringmethods in several aspects. First, the present monitoring methods canavoid any invasive procedures and this is particularly advantageous forhuman clinical uses. Second, the present monitoring methods offer invivo, real-time and continuous monitor and analysis of gene expressionin plants or animals, which cannot be accomplished using the currentlyavailable monitoring methods. Third, the present monitoring methods arequick and easily amenable to automation, which are important formonitoring large number of gene expression simultaneously. Since manydiseases or disorders involve abnormal gene expression of more thangene, the present monitoring methods are particularly suitable for theprognosis and diagnosis of these diseases or disorders. Besidesprognosis or diagnosis, if expression of certain genes is a goodindicator of tissue or organ health or functionality, the presentmonitoring methods can also be used in monitoring the health orfunctionality of these tissues or organs without any invasiveprocedures.

[0071] 1. Methods for Delivering The Nucleic Acids into theMulti-Cellular Organism

[0072] The nucleic acids encoding a fluorophore operatively linked tothe promoter of a gene whose expression is to be analyzed can be a DNAor a RNA. Such nucleic acids can be delivered into the body of themulti-cellular organism by any methods known in the art.

[0073] For example, if the host multi-cellular organism is an animal,the DNA or RNA sequence can be delivered to the interstitial space oftissues of the animal body, including those of muscle; skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers or organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation of the lymph fluid ofthe lymphatic channels.

[0074] The DNA or RNA sequence can be conveniently delivered byinjection into the tissues comprising these cells. They are preferablydelivered to and expressed in persistent, non-dividing cells which aredifferentiated, although delivery and expression can be achieved innon-differentiated or less completely differentiated cells, such as, forexample, stem cells of blood or skin fibroblasts.

[0075] In a specific embodiment, the DNA or RNA sequence is delivereddirectly to a tissue of the host animal. Preferably, the DNA or RNAsequence is delivered directly to muscle, skin or mucous membrane.Delivery to the interstitial space of muscle tissue is preferred becausemuscle cells are particularly competent in their ability to take up andexpress polynucleotides.

[0076] The DNA or RNA sequence can be delivered directly to a tissue ofthe host animal by injection, by gene gun technology or by lipidmediated delivery technology. The injection can be conducted via aneedle or other injection devices. The gene gun technology is disclosedin U.S. Pat. No. 5,302,509 and the lipid mediated delivery technology isdisclosed in U.S. Pat. No. 5,703,055.

[0077] In still another specific embodiment, the DNA or RNA sequence isdelivered to a cell of host animal and said cell containing the DNA orRNA sequence is delivered to a suitable tissue of the host animal.Preferably, the DNA or RNA sequence is delivered to tail or portal veinof the host animal.

[0078] The DNA or RNA sequence can be delivered to the cells of the hostanimal by a number of methods (see generally Koprowski & Weiner, DNAvaccination/genetic vaccination, 1998. Springer-verlag BerlinHeidelberg) including Ca₃(PO₄)₂-DNA transfection (Sambrook et al.,Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring HarborPress, 1989), DEAE dextran-DNA transfection (Sambrook et al., MolecularCloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press, 1989),electroporation (e.g., protocols from Bio-Rad), transfection using“LIPOFECTIN”™ reagent (e.g., protocols from BRL-Life Science), gene guntechnology (U.S. Pat. No. 5,302,509), or viral gene delivery system(Kaplitt et al., Viral Vectors, Academic Press, Inc., 1995).

[0079] Gold-particle based gene gun delivery is disclosed in U.S. Pat.No. 5,302,509. In a specific embodiment, Bio-Rad helios gene gun systemis used in the DNA delivery. (BIO-RAD Inc. New England). The helios genegun is a convenient, hand-held device that provides rapid and directgene transfer in vivo. The device employs an adjustable, helium pulse tosweep DNA coated gold microcarriers from the inner wall of a smallplastic cartridge directly into the target cells. The tubing prepstationand tubing cutter provide a simple way to prepare 50 cartridge “bullets”at a time.

[0080] In a preferred embodiment, a nucleic acid encoding a fluorophoreoperatively linked to the promoter of the gene is delivered directly tothe organism. More preferably, the nucleic acid encoding a fluorophoreoperatively linked to the promoter of the gene is delivered to theorganism, or to a cell to be delivered to the organism, in a viralvector such as a viral vector derived from adenovirus or a lentivirus.

[0081] Any viral vectors known in the art can be used. For example,vectors derived from a parvovirus (U.S. Pat. Nos. 5,252,479 and5,624,820), a paramyxovirus such as simian virus 5 (SV5) (U.S. Pat. No.5,962,274), a retrovirus such as HIV (U.S. Pat. Nos. 5,753,499 and5,888,767), and a baculovirus such as a nuclear polyhedrosis virus (U.S.Pat. No. 5,674,747) can be used. Preferably, a vector derived fromadenovirus can be used (U.S. Pat. Nos. 5,670,488, 5,817,492, 5,820,868,5,856,152, 5,981,225).

[0082] U.S. Pat. No. 5,670,488 discloses an adenoviral vector comprisingan adenovirus genome from which one or more of the E4 open readingframes has been deleted, but retaining sufficient E4 sequences topromote virus replication in vitro, and additionally comprising a DNAsequence of interest operably linked to expression control sequences andinserted into said adenoviral genome.

[0083] U.S. Pat. No. 5,817,492 discloses a recombinant adenoviral vectorcomprising: two DNA sequences which serve as a substrate for arecombinase enzyme, an origin of replication which is operable in ananimal cell, a promoter, a foreign gene and a poly(A) sequence, whereinsaid origin of replication, promoter, foreign gene and poly(A) sequenceare located between the two DNA sequences, and wherein said vectorcontains an E1A gene region deletion.

[0084] U.S. Pat. No. 5,820,868 discloses a live recombinant bovineadenovirus vector (BAV) wherein a part or all of the E3 multiple genecoding region is replaced by a heterologous nucleotide sequence encodinga foreign gene or fragment thereof. It also discloses a live recombinantbovine adenovirus vector (BAV) wherein part or all of the E3 multiplegene coding region is replaced by a heterologous nucleotide sequenceencoding a foreign gene or fragment thereof and wherein saidheterologous nucleotide sequence is optionally under the control of apromoter not normally associated with either said foreign gene or thebovine adenovirus genome.

[0085] U.S. Pat. No. 5,856,152 discloses a hybrid viral vectorcomprising: (a) adenovirus sequences comprising the adenovirus 5′ and 3′cis-elements necessary for replication and virion encapsidation; and (b)adeno-associated virus sequences comprising the 5′ and 3′ ITRs of anadeno-associated virus, said adeno-associated virus sequences flanked bythe adenoviral sequences of (a); and (c) a selected gene operativelylinked to regulatory sequences which direct its expression in a targetcell, said gene and regulatory sequences flanked by the adeno-associatedvirus sequences of (b).

[0086] U.S. Pat. No. 5,981,225 discloses a gene transfer vectorconsisting essentially of, in 5′ to 3′ orientation, the followingelements: (i) a first adenovirus inverted terminal repeat, (ii) anadenoviral VAI gene and/or VAII gene, (iii) a gene foreign toadenovirus, wherein said gene is operably linked to a promoterfunctional in adenovirus target cells, and (iv) a second adenovirusinverted terminal repeat, wherein the order of elements (ii) and (iii)may be reversed; and wherein one or both of element (i) and element (iv)additionally comprise an adenovirus packaging signal, and wherein saidvector is incapable of producing, in vitro, recombinant adenovirus virusparticles which have encapsidated therein said vector unless said vectoris co-transfected or co-infected into adenovirus host cells withadenovirus genomic DNA or adenovirus particles containing adenovirusgenomic DNA, respectively.

[0087] In another preferred embodiment, cells containing a nucleic acidencoding a fluorophore operatively linked to the promoter of the geneare delivered to the organism. More preferably, the cells are deliveredto the organism via a surgical procedure such as direct implantation bysurgical orthotopic implantation (SOI) at a desired site (see e.g.,Chang et al., Anticancer Res., 19(5B):4199 (1999); and An et al.,Prostate, 34(3):169-74 (1998)).

[0088] It will be understood, that by introducing a nucleic acidmolecule wherein a promoter is coupled to a nucleotide sequence encodinga fluorescent reporter gene, the introduced nucleic acid molecule can beused as a surrogate for the endogenous promoter. Thus, if the endogenousgene is over-expressed or under-expressed in the context of a particularcondition, the behavior of the introduced construct will mimic that ofthe endogenous promoter. It is not necessary that the reporter-encodingnucleotide sequence be operably linked only to a promoter; thenucleotide sequence encoding reporter may be introduced into thenucleotide sequence encoding the protein normally under control of thepromoter or coupled to another protein. Any method of operably linkingthe nucleotide sequence encoding reporter to the control sequences forthe gene whose expression is to be monitored falls within the scope ofthe invention.

[0089] It will be seen that there are a number of ways to introduce thisconstruct. First, the nucleic acid comprising the reporter encodingnucleotide sequence operably linked to the control sequences/promoter ofinterest can be introduced to the multicellular organism by directinjection, but preferably using a viral vector, such as a adenoviralvector or a lentiviral vector. Since the introduced construct is notendogenous, the expression of this construct essentially functions as asurrogate for the endogenous gene. That is, the same influences whichinfluence the endogenous gene will also influence the introducedconstruct. Thus, the conclusions reached by observing the expression ofthe construct, including the effects of various treatments on suchexpression, can be extrapolated to, and are equally valid for, thecounterpart endogenous gene.

[0090] Second, the reporter encoding nucleotide sequence could beintroduced into the cells of a particular tissue by targeting to thepromoter to be studied and inserted using position-specific techniques,such as homologous recombination. When this method is used, theexpression of the endogenous promoter can be observed directly as wellas can the effect of various treatments thereon.

[0091] Third, a construct such as those described for the first methodcan be provided to embryonic tissue to obtain transgenic organisms wherethe reporter construct is itself endogenous, see, for example, Fukumura,D., et al., Cell (1998) 94:715-725, incorporated herein by reference,which describes transgenic mice which use GFP as a reporter for VEGFpromoter activity.

[0092] Techniques for all three methods are well known in the art.

[0093] 2. Fluorophores

[0094] Any fluorophores known in the art can be used in the presentmethods. In a preferred embodiment, the fluorophore operatively linkedto the promoter of a gene is a humanized fluorophore. Also preferably,the fluorophore is a green fluorescent protein (GFP), a blue fluorescentprotein (BFP) and a red fluorescent protein (RFP). More preferably, theGFP is the humanized hGFP—S65T.

[0095] The native gene encoding GFP has been cloned from thebioluminescent jellyfish Aequorea Victoria (Morin et al., J CellPhysiol., 77:313-318 (1972)). The availability of the gene has made itpossible to use GFP as a marker for gene expression. GFP itself is a 283amino acid protein with a molecular weight of 27 kD. It requires noadditional proteins from its native source nor does it requiresubstrates or cofactors available only in its native source in order tofluoresce (Prasher et al., Gene, 111:229-233 (1992); Yang et al., NatureBiotechnol., 14:1252-1256 (1996); and Cody et al., Biochemistry,32:1212-1218 (1993)). Mutants of the GFP gene have been found useful toenhance expression and to modify excitation and fluorescence. GFP-S65T(wherein serine at 65 is replaced with threonine) is particularly usefulin the invention method and has a single excitation peak at 490 nm.(Heim et al., Nature, 373:663-664 (1995)); and U.S. Pat. No. 5,625,048).Other mutants have also been disclosed by Delagrade et al.,Biotechnology, 13:151-154 (1995); Cormack et al., Gene, 173:33-38(1996); and Cramer et al. Nature Biotechnol., 14:315-319 (1996).Additional mutants are also disclosed in U.S. Pat. No. 5,625,048. Bysuitable modification, the spectrum of light emitted by the GFP can bealtered. Thus, although the term “GFP” is used in the presentapplication, the proteins included within this definition are notnecessarily green in appearance. Various forms of GFP exhibit colorsother than green and these, too, are included within the definition of“GFP” and are useful in the methods and materials of the invention. Inaddition, it is noted that green fluorescent proteins falling within thedefinition of “GFP” herein have been isolated from other organisms, suchas the sea pansy, Renilla reriformis. Any suitable and convenient formof the GFP gene can be used in the invention. Techniques for labelingcells in general using GFP are disclosed in U.S. Pat. No. 5,491,084(supra).

[0096] Other GFP, BFP and RFP can be used in the present methods. Forinstances, the green fluorescent proteins encoded by nucleic acids withthe following GenBank accession Nos. can be used: U47949 (AGP1); U43284;AF007834 (GFPuv); U89686 (Saccharomyces cerevisiae synthetic greenfluorescent protein (cox3::GFPm-3) gene); U89685 (Saccharomycescerevisiae synthetic green fluorescent protein (cox3::GFPm) gene);U87974 (Synthetic construct modified green fluorescent protein GFP5-ER(mgfp5-ER)); U87973 (Synthetic construct modified green fluorescentprotein GFP5 (mgfp5)); U87625 (Synthetic construct modified greenfluorescent protein GFP-ER (mfgp4-ER)); U87624 (Synthetic constructgreen fluorescent protein (mgfp4) mRNA)); U73901 (Aequorea victoriamutant 3); U50963 (Synthetic); U70495 (soluble-modified greenfluorescent protein (smGFP)); U57609 (enhanced green fluorescent proteingene); U57608 (enhanced green fluorescent protein gene); U57607(enhanced green fluorescent protein gene); U57606 (enhanced greenfluorescent protein gene); U55763 (enhanced green fluorescent protein(egfp); U55762 (enhanced green fluorescent protein (egfp); U55761(enhanced green fluorescent protein (egfp); U54830 (Synthetic E. coliTn3-derived transposon green fluorescent protein (GF); U36202; U36201;U19282; U9279; U19277; U19276; U19281; U19280; U19278; L29345 (Aequoreavictoria); M62654 (Aequorea victoria); M62653 (Aequorea victoria);AAB47853 ((U87625) synthetic construct modified green fluorescentprotein (GFP-ER)); AAB47852 ((U87624) synthetic construct greenfluorescent protein).

[0097] Similarly, the blue fluorescent proteins encoded by nucleic acidswith the following GenBank accession Nos. can be used: U70497(soluble-modified blue fluorescent protein (smBFP); 1BFP (blue variantof green fluorescent protein); AAB16959 (soluble-modified bluefluorescent protein).

[0098] Also similarly, the red fluorescent proteins encoded by nucleicacids with the following GenBank accession Nos. can be used: U70496(soluble-modified red-shifted green fluorescent protein (smRSGFP);AAB16958 (U70496) soluble-modified red-shifted green fluorescentprotein).

[0099] A fluorophore that changes color with time is reported byTeiskikh, A., et al., Science (2000) 290:1585-1588, incorporated hereinby reference. This permits tracing time dependent expression.

[0100] 3. Multi-Cellular Organisms

[0101] The present methods can be used in monitoring gene expression inany suitable multi-cellular organisms. In a preferred embodiment, themulti-cellular organism to be analyzed is a plant or an animal,including a transgenic animal. More preferably, the animal is a mammalincluding a human. Animals that can be analyzed with the presentmonitoring emthods include, but are not limited to, mice, rats, rabbits,cats, dogs, pigs, cows, ox, sheep, goats, horses, monkeys and othernon-human primates.

[0102] 4. Tissue or Organ Specific Gene Expression

[0103] The present methods can be used in monitoring expression of genesthat are expressed in a tissue or organ specific manner. The presentmethods can be used in monitoring health and/or functionality of tissuesand/or organs if expression pattern of certain genes are associated withhealth and/or functionality of these tissues and organs. Preferably, thegene to be monitored is expressed in connective, epithelium, muscle ornerve tissue. Also preferably, the gene to be monitored is expressed inan accessory organ of the eye, annulospiral organ, auditory organ,Chievitz organ, circumventricular organ, Corti organ, critical organ,enamel organ, end organ, external female gential organ, external malegenital organ, floating organ, flower-spray organ of Ruffini, genitalorgan, Golgi tendon organ, gustatory organ, organ of hearing, internalfemale genital organ, internal male genital organ, intromittent organ,Jacobson organ, neurohemal organ, neurotendinous organ, olfactory organ,otolithic organ, ptotic organ, organ of Rosenmüller, sense organ, organof smell, spiral organ, subcommissural organ, subfomical organ,supernumerary organ, tactile organ, target organ, organ of taste, organof touch, urinary organ, vascular organ of lamina terminalis, vestibularorgan, vestibulocochlear organ, vestigial organ, organ of vision, visualorgan, vomeronasal organ, wandering organ, Weber organ and organ ofZuckerkandl. More preferably, the gene to be monitored is expressed inan internal animal organ such as brain, lung, liver, spleen, bonemarrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney,gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, gland, internal blood vessels, etc.

[0104] In other embodiments, the fluorophore, e.g., GFP, BFP or RFP, canbe operatively linked to the following animal transcriptional controlregions that exhibit tissue specificity to monitor these tissue specificgene expressions in animals: elastase I gene control region which isactive in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984);Ornitz et al., Cold Spring Harbor Symp. Qziant. Biol. 50:399-409 (1986);MacDonald, Hepatology 7:425-515 (1987)); insulin gene control regionwhich is active in pancreatic beta cells (Hanahan et al., Nature315:115-122 (1985)), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams etal., Nature 318:533-538 (1985); Alexander et al., Mol. Cell Biol.7:1436-1444 (1987)), mouse mammary tumor virus control region which isactive in testicular, breast, lymphoid and mast cells (Leder et al.,Cell 45:485-495 (1986)), albumin gene control region which is active inliver (Pinckert et al., Genes and Devel. 1:268-276 (1987)),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science235:53-58 1987)), alpha-1 antitrypsin gene control region which isactive in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), betaglobin gene control region which is active in myeloid cells (Mogram etal., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)),myelin basic protein gene control region which is active inoligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712(1987)), myosin light chain-2 gene control region which is active inskeletal muscle (Sani, Nature 314:283-286 (1985)), and gonadotrophicreleasing hormone gene control region which is active in gonadotrophs ofthe hypothalamus (Mason et al., Science 234:1372-1378 (1986)).

[0105] 5. Tumor or Cancer Associated Gene Expression

[0106] The present methods can be used in monitoring expression of genesthat are specifically expressed in tumors or cancers. Preferably, thegene to be analyzed is a tumor or cancer associated gene such as anoncogene or a tumor suppressor gene. For instance, the expression of theoncogenes listed in the following Table 1 can be monitored by thepresent methods. TABLE 1 Oncogenes and tumor viruses Acronym VirusSpecies Tumor origin Comments abl Abelson leukemia Mouse ChronicTyrPK(src) myelogenous leukemia erbA Erythroblastosis Chicken Homologyto human glucocorticoid receptor erbB Erythroblastosis Chicken TryPKEGF/TGFc receptor ets E26 myeloblastosis Chicken Nuclear fes (fps)^(a)Snyder-Thellen Cat TryPK(src) sarcoma Gardner-Arnstein sarcoma fgrGardner-Rasheed Cat TyrPK(src) sarcoma fins McDonough sarcoma Cat TyrPKCSF-1 receptor fps (fes)^(a) Fujinami sarcoma Chicken TyrPK(src) fos FBJosteosarcoma Mouse Nuclear, TR hst NVT Human Stomach tumor FGF homologueintl NVT Mouse MMTV-induced Nuclear, TR carcinoma int2 NVT MouseMMTV-induced FGF homologue carcinoma jun ASV17 sarcoma Chicken Nuclear,TR hit Hardy-Zuckerman 4 Cat TyrPK GFR L sarcoma B-lym NVT ChickenBursal lymphoma mas NVT Human Epidermoiod Potentiates response carcinomato angiotensin II met NVT Mouse Osteosarcoma TyrPK GFR L mil (raf)^(b)Mill Hill 2 acute Chicken Ser/ThrPK leukemia mos Moloney sarcoma MouseSer/ThrPK myb Myeloblastosis Chicken Leukemia Nuclear, TR myc MC29Chicken Lymphomas Nuclear TR myelocytomatosis N-myc NVT HumanNeuroblastomas Nuclear neu (ErbB2) NVT Rat Neuroblastoma TryPK GFR L ral(mil)^(b) 3611 sarcoma Mouse Ser/ThrPK Ha-ras Harvey murine Rat Bladder,mammary GTP-binding sarcoma and skin carcinomas Ki-ras Kirsten murineRat Lung, colon GTP-binding sarcoma carcinomas N-ras NVT HumanNeuroblastomas GTP-binding leukaemias rel Reticuloendothe-liosis Turkeyros UR2 Chicken TyrPK GFR L sis Simian sarcoma Monkey One chain of PDGFsrc Rous sarcoma Chicken TyrPK ski SKV770 Chicken Nuclear trk NVT HumanColon carcinoma TyrPK GFR L yes Y73, Esh sarcoma Chicken TyrPK(src)

[0107] Similarly, the expression of the following tumor suppressor genescan be monitored by the present methods: p16, p21, p27, p53, RB, WT-1,DCC, NF-1 and APC.

[0108] Since abnormally high level of oncogene expression and abnormallylow expression of tumor suppressor gene are often good indicators ofoncogenesis, the present methods can be used in prognosis or diagnosisof cancer, in monitoring the development of oncogenesis and inevaluating the efficacy of the cancer therapy.

[0109] C. Methods to Evaluate a Candidate Protocol or Drug for TreatingDisease or Disorder

[0110] Since the method of the invention evaluates gene expression withregard to particular control sequences, the effect of various compounds,treatments (such as irradiation) or other perturbations of the geneticenvironment can be evaluated for their effect on expression using themethods of the invention. Thus, gene toxic agents, for example, can beidentified.

[0111] In a specific embodiment, a method to evaluate a candidateprotocol or drug for treating a disease or disorder is provide herein,which method comprises: a) administering said protocol or drug to anon-human mammalian subject which expresses a fluorophore under thedirection of a promoter of a gene associated with a disease or disorder,and determining the expression of said promoter via observing thepresence, absence or intensity of the fluorescence generated by saidfluorophore at various locations in said mammalian subject by whole-bodyexternal fluorescent optical imaging; b) determining the expression ofsaid promoter, via observing the presence, absence or intensity of thefluorescence generated by said fluorophore at various locations in saidmammalian subject by whole-body external fluorescent optical imaging, ina control non-human mammalian subject which expresses said fluorophoreunder the direction of said promoter of said gene; and c) comparing theexpression of said promoter determined in steps a) and b), wherein theexpression determined in step a) is different from that in step b)identifies said protocol or drug as effective in treating the disease ordisorder.

[0112] In a preferred embodiment, overexpression of the gene isassociated with the disease or disorder and the expression determined instep a) is lower than that in step b) identifies said protocol or drugas effective in treating the disease or disorder.

[0113] In another preferred embodiment, underexpression of the gene isassociated with the disease or disorder and the expression determined instep a) is higher than that in step b) identifies said protocol or drugas effective in treating the infection.

[0114] In still another preferred embodiment, the non-human mammaliansubject which expresses a fluorophore under the direction of a promoterof a gene associated with a disease or disorder is produced bydelivering a nucleic acid encoding the fluorophore operatively linked tothe promoter, or a cell containing the nucleic acid, to the non-humanmammalian subject (see Section B supra).

[0115] Any non-human mammalian subject can be used in the presentscreening methods. Preferably, the non-human mammalian subject used inthe screening is a well established laboratory animal such as a mice, arabbit or a non-human primate. Also preferably, the non-human mammaliansubject used in the screening is an infectious disease animal model.Still preferably, the non-human mammalian subject used in the screen isa transgenic animal.

[0116] Any fluorophores known in the art, including the ones describedin Section B, can used in the present screening methods. In a preferredembodiment, the fluorophore used in the screening is s a greenfluorescent protein (GFP), a blue fluorescent protein (BFP) or a redfluorescent protein (RFP).

[0117] The present methods can be used to screen candidate protocols ordrugs for treating any known diseases or disorders. In a preferredembodiment, the diseases or disorders to be screened against arecancers, immune system diseases or disorders, metabolism diseases ordisorders, muscle and bone diseases or disorders, nervous systemdiseases or disorders, signal diseases or disorders and transporterdiseases or disorders.

[0118] In yet another preferred embodiment, the non-human mammaliansubject expresses a fluorophore under the direction of a promoter of aninfectious organism and the expression determined in step a) is lowerthan that in step b) identifies said protocol or drug as effective intreating infection caused by the infectious organism.

[0119] The non-human mammalian subject used in the screening may be aninfectious disease animal model.

[0120] The infectious organism screened against may be a fungus such asa yeast, a bacterium such as an eubacteria or an archaebacteria, or avirus such as a Class I virus, a Class II virus, a Class III virus, aClass IV virus, a Class V virus or a Class VI virus.

[0121] Any substances can be screened using the present screeningmethods for finding drug candidates for treating infection. In apreferred embodiment, a combinatorial library is used in the screeningassays. Methods for synthesizing combinatorial libraries andcharacteristics of such combinatorial libraries are known in the art(See generally, Combinatorial Libraries: Synthesis, Screening andApplication Potential (Cortese Ed.) Walter de Gruyter, Inc., 1995;Tietze and Lieb, Curr. Opin. Chem. Biol., 2(3):363-71 (1998); Lam,Anticancer Drug Des., 12(3):145-67 (1997); Blaney and Martin, Curr.Opin. Chem. Biol., 1(1):54-9 (1997); and Schultz and Schultz,Biotechnol. Prog., 12(6):729-43 (1996)).

[0122] If the infection is caused by bacteria, known antibiotics can bescreened using the present screening methods for finding a suitable drugcandidate. Preferably, the antibiotics to be screened areaminoglycosides (e.g., streptomycin, gentamicin, sisomicin, tobramycin,amicacin), ansamycins (e.g., rifanycin), antimycotics polyenes (e.g.,nystatin, pimaricin, amphotericin B., pecilocin), benzofuran derivatives(e.g., griseofulvin), β-lactam antibiotics penicillins (e.g., penicillinG and its derivatives, oral penicillins, penicillinase-fixed penicillinbroad-spectrum penicillins, penicillins active against Proteus andPseudomonas), cephalosporins (e.g., cephalothin, cephaloridine,cephalexin, cefazolin, cefotaxime), chloramphenicol group (e.g.,chloramphenicol, thiamphenicol, azidamphenicol), Imidazole fluconazole,itraconazole, linosamides (e.g., lincomycin, clindamycin), macrolides(e.g., azithromycin, erythromycin, oleandomycin, spiramycin,clarithromycin), peptides, peptolides, polypeptides (e.g., polymyxin Band E, bacitracin, tyrothricin, capreomycin, vancomycin), quinolones(e.g., nalidixic acid, ofloxacin, ciprofloxacin, norfioxin),tetracyclines (e.g., tetracycline, oxytetracycline, minocycline,doxycycline) and other antibiotics (e.g., phosphomycin, fusidic acid):

[0123] D. Methods to Screen for Gene Expression Modulators andRegulators

[0124] The above-described screening methods can also be used toidentify gene expression modulators, i.e., trans-acting substances thatmodulate the expression of a target gene in a multi-cellular organism,or regulators, i.e., cis-acting genes of a multi-cellular organism thatregulate the expression of the target gene. Besides for identifyingdisease or disorder treatment protocols or drugs, the screening methodsdescribed herein have wide applications in industrial, agricultural,environmental protection and many other fields. For example, transgenicanimals such as transgenic cows are commercially used. It is desirableto find a suitable substance that increases the expression of thetransgene and such substance can be added to the animal feed. Similarly,it is desirable to find and modify gene(s) within the transgenic cowthat enhances the expression of the target transgene.

[0125] Once it is decided that alteration of the expression level of atarget gene is desirable, a fluorophore can be operatively linked to thepromoter, or other transcriptional control region, of the target geneand be expressed in a multi-cellular organism. Then, the multi-cellularorganism expressing the fluorophore can be treated with a test substanceto identify which substance modulates the fluorophore expression.Alternatively, the multi-cellular organism expressing the fluorophoreitself can be mutagenized to identify genes within itself that alter thefluorophore expression. These screening principles have long been usedto identify cis- or trans-acting regulators of gene expression inunicellular organisms such as bacteria or yeast. However, due to thelack of quick and simple screening methods, such screening areimpractical for multi-cellular organisms such as plants and animals. Thewhole-body external optical imaging of gene expression disclosed hereinmakes such screening or mutant-haunt practical for multi-cellularorganisms.

[0126] In a specific embodiment, a method to screen for a modulator ofthe expression of a gene in a multi-cellular organism is providedherein, which method comprises: a) administering a test substance to anon-human multi-cellular organism which expresses a fluorophore underthe direction of a promoter of a gene, and determining the expression ofsaid promoter via observing the presence, absence or intensity of thefluorescence generated by said fluorophore at various locations in saidmulti-cellular organism by whole-body external fluorescent opticalimaging; b) determining the expression of said promoter, via observingthe presence, absence or intensity of the fluorescence generated by saidfluorophore at various locations by whole-body external fluorescentoptical imaging, in a control multi-cellular organism which expressessaid fluorophore under the direction of said promoter of said gene; andc) comparing the expression of said promoter determined in steps a) andb), wherein the expression determined in step a) is different from thatin step b) identifies said test substance as a modulator of said geneexpression. Preferably, the promoter is an endogenous promoter of themulti-cellular organism.

[0127] In another specific embodiment, a method to screen for amulti-cellular organism that expresses a gene at an altered level isprovided herein, which method comprises: a) administering amutation-inducing agent or treatment to a non-human multi-cellularorganism which expresses a fluorophore under the direction of a promoterof a gene, and determining the expression of said promoter via observingthe presence, absence or intensity of the fluorescence generated by saidfluorophore at various locations in said multi-cellular organism bywhole-body external fluorescent optical imaging; b) determining theexpression of said promoter, via observing the presence, absence orintensity of the fluorescence generated by said fluorophore at variouslocations by whole-body external fluorescent optical imaging, in anuntreated control multi-cellular organism which expresses saidfluorophore under the direction of said promoter of said gene; and c)comparing the expression of said promoter determined in steps a) and b),wherein the expression determined in step a) is different from that instep b) identifies a multi-cellular organism that expresses said gene atsaid altered level. Preferably, the mutation-inducing agent or treatmentcauses a mutation in germ-line cells of the multi-cellular organism sothat the desired mutation is stably-transferable to offspring of themulti-cellular organism.

[0128] In addition, the various protocols described in the art for “BigBlue” transgenic mice can be utilized in the system of the invention.

[0129] The following examples are included for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLE 1 Visualization of Gene Expression in Various Tissues usingAdenovirus

[0130] Four six-week-old male of female nude/nude, nude/+, or C57BL/6mice were used. All animal studies were conducted in accordance with theprinciples and procedures outlined in the National Institute of HealthGuide for the Care and Use of Animals under assurance number A3873-1.Mice were fed with autoclaved laboratory rodent diet (Tecklad LM-485,Western Research Products, Orange, Calif.).

[0131] The vector employed was adenorival (vAd) vector AdCMV5GFPAEl/AE3[vAd-green fluorescent protein (GFP)] (Quantum, Montreal, Canada), whichexpresses enhanced GFP and the ampicillin resistance gene.

[0132] This vector was provided to various tissues to visualizeexpression of the CMV promoter in these tissues. Expression of reporterunder control of any desired promoter can be visualized by suitablemodification of this vector, as described above.

[0133] Liver: After exposure of the portal vein following an uppermidline abdominal incision, total volume of 100 μl (8×10¹⁰ pfu/ml)vAd-GFP per mouse were injected in the portal vein using a 1 ml 39G1latex-free syringe (Becton Dickinson, Franklin Lakes, N.J.). Thepuncture hole of portal vein was pressed for about 10 seconds withsterile cotton to stop any bleeding. The incision in the abdominal wallwas closed with a 7-0 surgical suture in one layer. The animals werekept under Ketamine anesthesia during surgery. All procedures of theoperation described above were performed with a 7× magnificationmicroscope (Leica MZ6, Nussloch, Germany). Animals were kept in abarrier facility under HEPA filtration.

[0134] Brain: The parietal bone of the skull was exposed after an uppermidline scalp incision. Twenty microliters containing 8×10¹⁰plaque-forming units (pfu)/ml vAd-GFP per mouse was injected in thebrain by using a 1-ml 27G1/2 latex-free syringe (Becton Dickinson). Thepuncture hole in the skull was plugged with bone wax. The incision inthe scalp was closed with a 7-0 surgical suture in one layer. Theanimals were kept under isofluorane anesthesia during surgery.

[0135] Pancreas: The pancreas was exposed after an upper midlineabdominal incision. One-hundred microliters containing 8×10¹⁰ pfu/mlvAd-GFP per mouse was injected in the pancreas by using a 1-ml 30G_(1/2)latex-free syringe (Becton Dickinson). The puncture hole was pressed forabout 10 sec with sterile cotton for hemostasis. The incision was closedwith a 7-0 surgical suture in one layer. The animals were kept underKersel anesthesia during surgery. All procedures of the operationdescribed above were performed with a ×7 magnification stereomicroscope.

[0136] Prostate: The bladder and prostate were exposed after a lowermidline abdominal incision. Thirty microliters containing 8×10¹⁰ pfu/mlvAd-GFP per mouse was injected in the prostate by using a 1-ml 30G_(1/2)latex-free syringe (Becton Dickinson). The puncture hole in the prostatewas pressed for about 10 sec with sterile cotton for hemostasis. Theincision in the abdominal wall was closed with a 6-0 surgical suture inone layer. The animals were kept under isofluorane anesthesia duringsurgery. All procedures of the operation described above were performedwith a ×7 magnification stereo miscroscope.

[0137] Bone Marrow: For bone marrow injection, animals were anesthetizedby inhalation of isofluorane. The skin on the hind leg was opened with a1-cm incision to expose the tibia. A 27-gauge needle with latex-freesyringe (Becton Dickinson) then was inserted in the bone marrow cavity.A total volume of 20 ul (8×10¹⁰ pfu/ml) vAd-GFP per mouse was injectedinto the bone marrow cavity. The puncture hole in the bone was pluggedwith bone wax, and the incision was closed with a 6-0 surgical suture.

[0138] Visualization: For visualization at high magnification, Leicafluorescence stereo microscope, model LZ12, equipped with a 50-W mercurylamp, was used. Selective excitation of GFP was produced through aD425/60 band-pass filter and 470 DCXR dichroic mirror. Emittedfluorescence was collected through a long-pass filter GG475 (ChromaTechnology, Brattleboro, Vt.) on a Hamamatsu C5810-3-chip cooled colorcharge-coupled device camera (Hamamatsu Photonics Systems, Bridgewater,N.J.). Images were processed for contrast and brightness and analyzedwith the use of IMAGE PRO PLUS 3.1 software (Media Cybernetics, SilverSprings, Md.). Images of 1,024×724 pixels were captured directly on anIBM PC or continuously through video output on a high-resolution SonyVCR model SLV-R-1000 (Sony, Tokyo).

[0139] Imaging at lower magnification that visualized the entire animalwas carried out in a light box illuminated by blue light fiber optics(Lightools Research, Encinitas, Calif.) and imaged by using thethermoelectrically cooled color charge-coupled device camera, asdescribed above.

[0140] Quantitation: The intensity of GFP fluorescence is measured toaccount for variations in the exciting illumination with time and acrossthe imaging area. These factors are corrected for by using the intrinsicred fluorescence of mouse skin as a base line to correct the increaseover intrinsic green fluorescence caused by GFP. This can be donebecause there is relatively little red luminence in the GFP radiance.Consequently, the green fluorescence was calculated relative to redbased on red and green channel composition in the skin image. A ratio(γ) of green to red channels was determined for each pixel in the imageof skin without and with GFP. Values of γ for mouse skin throughout theimage in the absence of GFP were fairly constant, varying between 0.7and 1.0. The contribution of GFP fluorescence from within the animalincreased the green component relative to red, which was reflected inhigher γ values. The total amount of GFP fluorescence was approximatedby multiplying the number of pixels in which value γ was higher than 1times the γ value of each pixel. Such a product roughly corresponds tothe integral GFP fluorescence [I′_(GFP)] above the maximum value of γfor skin without GFP. The number of pixels in mouse skin images with γvalue >1.0 without GFP was less than 0.02% and increased with GFPexpression. The value of [I′_(GFP)] is shown as a function of time aftervirus injection in FIGS. 1A and 1B for brain and liver respectively.

[0141] Images of the various organs were compared when taken at highmagnification on live intact animals or similar organs viewed directlyafter death and dissection. The images show the distribution of geneexpression in the various organs. In all cases, the images madeexternally are similar to those of the exposed organs.

[0142] When the live animal was viewed in a light box, it was alsopossible to monitor the expression of the gene, thus permitting a realtime observation of the living animal and expression as it occurs inthis animal. For example, a light box determination of expression of theGFP in nude mouse liver taken at 72 hours clearly shows this result.Similar results are observed in the nude mouse brain 24 hours after genedelivery. The method is quite sensitive in that the intensity of GFPfluorescence in the mouse liver at a depth of 0.8 mm under the skin wasabout 25% of that of the exposed organ. Gene expression is externallymeasurable if the average fluorescence of the GFP expressing organs isat least 20% above the average fluorescence of the surrounding skin, andat maximal level of GFP expression, the intensity in the liver exceededmore than 100 times the back dorsal and abdominal skin fluorescence.

EXAMPLE 2 Visualization of Genes Using Lentiviral Vectors

[0143] Lentiviral vectors have been shown to transduce a broad spectrumof non-dividing cells in vitro, such as neurons, retina, liver, muscleand hematopoietic stem cells (see, for example, Naldini, L. et al.,Science (1996) 272:263-267; Kafri T. et al.; Nat. Genet (1997)17:314-317; Takahashi, M. et al., J. Virol (1999) 73:7812-7816; Miyoshi,H. et al. Science (1999) 283:682-686). Although it has been reportedthat hepatocytes are refractory to lentiviral transduction unless theyprogress into the cell cycle (Park, F. et al. Nat. Genet (2000)24:49-52), it is shown below that lentiviral gene delivery to the liverfor expression visualization is practical.

[0144] A lentiviral vector based on HIV1 designated GFP-LV was used.This vector contains a self-inactivating mutation in the U-3 region, apost-transcriptional element, and an internal CMV promoter. It alsocontains cppt, the central polypurine tract derived from HIV-pol and awoodchuck hepatitis virus post-transcriptional element (WPRE). A diagramof this vector is shown in FIG. 2A.

[0145] The vector GHP-LV at 1×10⁹ IU was injected into the portal veinof nude mice; (Hsd:asymic nude-nu). Six (6) days after injection greenfluorescence was testable in the liver using in-vivo fluorescenceoptical imaging, as shown in FIG. 2B. At day 21, all lobes of the liverof the mice injected with this vector exhibited a homogeneous greenfluorescence.

[0146] GHP-LV at 1×10⁹ IU was also injected intraperitoneally and thismethod also resulted in a high level of transduction of liver andspleen.

[0147] Western Blot demonstrated dose dependence of GFP expression inthe range of 0.5-2.5×10⁹ IU. Vector integration in the liver 3 weeksafter injection was demonstrated by PCR.

[0148] Confirmation that the transduced cells were not rapidly dividingwas achieved by administering 5′bromo-2′deoxyuridine (BrdU) 15 mgs/kg bydaily IP injections in order to label dividing cells. While the cells inthe duodenum showed high labelling, only about 3% of liver cells wereBrdU positive in either control or lentiviral-treated livers.

EXAMPLE 3 Additional Applications

[0149] In addition to the procedures exemplified in Examples 1 and 2,the methods of the invention may be used to monitor expression ofcontrol sequences that are regulated by the unfolded protein response(UPR) as described, for example, by Niwa, M., et al., Cell (1999)99:691-702, the contents of which are incorporated herein by reference.Another suitable target for study is the circadian rhythm controllinggenes which were studied using less convenient techniques by Yamaguchi,S., et al., Nature (2001) 409:684, incorporated herein by reference.

[0150] Since modifications will be apparent to those of skill in thisart, it is intended that this invention be limited only by the scope ofthe appended claims.

1. A method to monitor the expression of a gene, which method comprises:a) delivering to a multi-cellular organism a nucleic acid encoding afluorophore operatively linked to the promoter of a gene whoseexpression is to be analyzed or delivering a cell containing saidnucleic acid; and b) observing the presence, absence or intensity of thefluorescence generated by said fluorophore at various locations in saidorganism by whole-body external fluorescent optical imaging, whereby theexpression of said gene is monitored.
 2. The method of claim 1, whereina nucleic acid encoding a fluorophore operatively linked to the promoterof the gene is delivered to the organism.
 3. The method of claim 1,wherein the nucleic acid is comprised in a viral vector.
 4. The methodof claim 3, wherein the viral vector is derived from adenovirus.
 5. Themethod of claim 1, wherein a cell containing the nucleic acid isdelivered to the organism.
 6. The method of claim 5, wherein the cell isdelivered to the organism via a surgical procedure.
 7. The method ofclaim 6, wherein the cell is delivered to the organism via directimplantation by surgical orthotopic implantation (SOI) at a desiredsite.
 8. The method of claim 1, wherein the fluorophore is a humanizedfluorophore.
 9. The method of claim 1, wherein the fluorophore isselected from the group consisting of a green fluorescent protein (GFP),a blue fluorescent protein (BFP) and a red fluorescent protein (RFP).10. The method of claim 9, wherein the GFP is the humanized hGFP-S65T.11. The method of claim 1, wherein the multi-cellular organism is aplant or an animal.
 12. The method of claim 11, wherein the animal is amammal.
 13. The method of claim 12, wherein the mammal is selected fromthe group consisting of a mouse, a rat, a rabbit, a cat, a dog, a pig, acow, an ox, a sheep, a goat, a horse, a monkey and a non-human primate.14. The method of claim 11, wherein the animal is a transgenic animal.15. The method of claim 1, wherein the gene is expressed in a tissue ororgan specific manner.
 16. The method of claim 15, wherein the tissue isselected from the group consisting of connective, epithelium, muscle andnerve tissues.
 17. The method of claim 15, wherein the organ is selectedfrom the group consisting of brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, gland, and internal blood vessels.
 18. The method of claim 1,wherein the gene is a tumor or cancer associated gene.
 19. The method ofclaim 18, wherein the tumor or cancer associated gene is an oncogene ora tumor suppressor gene.
 20. A method to evaluate a candidate protocolor drug for treating a disease or disorder, which method comprises: a)administering said protocol or drug to a non-human mammalian subjectwhich expresses a fluorophore under the direction of a promoter of agene associated with said disease or disorder, and determining theexpression of said gene via observing the presence, absence or intensityof the fluorescence generated by said fluorophore at various locationsin said mammalian subject by whole-body external fluorescent opticalimaging; b) determining the expression of said gene, via observing thepresence, absence or intensity of the fluorescence generated by saidfluorophore at various locations in said mammalian subject by whole-bodyexternal fluorescent optical imaging, in a control non-human mammaliansubject which expresses said fluorophore under the direction of saidpromoter of said gene; and c) comparing the expression of said promoterdetermined in steps a) and b), wherein the expression determined in stepa) is different from that in step b) identifies said protocol or drug aseffective in treating the disease or disorder.
 21. The method of claim20, wherein overexpression of the gene is associated with the disease ordisorder and the expression determined in step a) is lower than that instep b) when said protocol or drug is effective in treating the diseaseor disorder.
 22. The method of claim 20, wherein underexpression of thegene is associated with the disease or disorder and the expressiondetermined in step a) is higher than that in step b) when said protocolor drug is effective in treating the infection.
 23. The method of claim20, wherein the disease or disorder is selected from the groupconsisting of a cancer, an immune system disease or disorder, ametabolism disease or disorder, a muscle and bone disease or disorder, anervous system disease or disorder, a signal disease or disorder and atransporter disease or disorder.
 24. The method of claim 20, wherein thepromoter is derived from an infectious organism.
 25. The method of claim20, wherein the non-human mammalian subject which expresses afluorophore under the direction of a promoter of the gene is produced bydelivering a nucleic acid encoding the fluorophore operatively linked tothe promoter of the gene, or delivering a cell containing the nucleicacid, to the non-human mammalian subject.
 26. The method of claim 20,wherein the non-human mammalian subject is selected from the groupconsisting of a mouse, a rat, a rabbit, a cat, a dog, a pig, a cow, anox, a sheep, a goat, a horse, a monkey and a non-human primate.
 27. Themethod of claim 24, wherein the non-human mammalian subject is aninfectious disease animal model.
 28. The method of claim 20, wherein thenon-human mammalian subject is a transgenic animal.
 29. The method ofclaim 20, wherein the fluorophore is selected from the group consistingof a green fluorescent protein (GFP), a blue fluorescent protein (BFP)and a red fluorescent protein (RFP).
 30. The method of claim 24, whereinthe infectious organism is selected from the group consisting of afungus, a bacterium and a virus.
 31. The method of claim 30, wherein thefungus is a yeast.
 32. The method of claim 30, wherein the bacterium isan eubacteria or an archaebacteria.
 33. The method of claim 30, whereinthe virus is selected from the group consisting of a Class I virus, aClass II virus, a Class III virus, a Class IV virus, a Class V virus anda Class VI virus.
 34. The method of claim 24, wherein the candidate drugto be screened is an antibiotic.
 35. The method of claim 1, wherein theexpression of more than one gene is monitored simultaneously.
 36. Themethod of claim 20, wherein more than one candidate protocol orcandidate drug is screened for simultaneously.
 37. A method to screenfor a modulator of the expression of a gene in a multi-cellularorganism, which method comprises: a) administering a test substance to anon-human multi-cellular organism which expresses a fluorophore underthe direction of a promoter of a gene, and determining the expression ofsaid promoter via observing the presence, absence or intensity of thefluorescence generated by said fluorophore at various locations in saidmulti-cellular organism by whole-body external fluorescent opticalimaging; b) determining the expression of said promoter, via observingthe presence, absence or intensity of the fluorescence generated by saidfluorophore at various locations by whole-body external fluorescentoptical imaging, in a control multi-cellular organism which expressessaid fluorophore under the direction of said promoter of said gene; andc) comparing the expression of said promoter determined in steps a) andb), wherein the expression determined in step a) is different from thatin step b) when said test substance modulates said gene expression. 38.The method of claim 37, wherein the promoter is an endogenous promoterof the multi-cellular organism.
 39. A method to screen for amulti-cellular organism that expresses a gene at an altered level, whichmethod comprises: a) administering a mutation-inducing agent ortreatment to a non-human multi-cellular organism which expresses afluorophore under the direction of a promoter of a gene, and determiningthe expression of said promoter via observing the presence, absence orintensity of the fluorescence generated by said fluorophore at variouslocations in said multi-cellular organism by whole-body externalfluorescent optical imaging; b) determining the expression of saidpromoter, via observing the presence, absence or intensity of thefluorescence generated by said fluorophore at various locations bywhole-body external fluorescent optical imaging, in an untreated controlmulti-cellular organism which expresses said fluorophore under thedirection of said promoter of said gene; and c) comparing the expressionof said promoter determined in steps a) and b), wherein the expressiondetermined in step a) is different from that in step b) when saidmulti-cellular organism expresses said gene at said altered level. 40.The method of claim 39, wherein the mutation-inducing agent or treatmentcauses a mutation in germ-line cells of the multi-cellular organism sothat the mutation is stably-transferable to offspring of themulti-cellular organism.